Abstract

Current subunit vaccines are incapable of inducing Ag-specific CD8+ T cell cytotoxicity needed for the defense of certain infections and for therapy of neoplastic diseases. In experimental vaccines, cytotoxic responses can be elicited by targeting of Ag into cross-presenting dendritic cells (DC), but almost all available systems use target molecules also expressed on other cells and thus lack the desired specificity. In the present work, we induced CD8+ T cell cytotoxicity by targeting of Ag to XCR1, a chemokine receptor exclusively expressed on murine and human cross-presenting DC. Targeting of Ag with a mAb or the chemokine ligand XCL1 was highly specific, as determined with XCR1-deficient mice. When applied together with an adjuvant, both vector systems induced a potent cytotoxic response preventing the outgrowth of an inoculated aggressive tumor. By generating a transgenic mouse only expressing the human XCR1 on its cross-presenting DC, we could demonstrate that targeting of Ag using human XCL1 as vector is fully effective in vivo. The specificity and efficiency of XCR1-mediated Ag targeting to cross-presenting DC, combined with its lack of adverse effects, make this system a prime candidate for the development of therapeutic cytotoxic vaccines in humans.

Introduction

Modern subunit vaccines stimulate the B cell immune system and induce neutralizing Abs. However, neutralizing Abs cannot be effective against pathogens that “hide” intracellularly (e.g., Plasmodium malariae), or that permanently change the antigenic nature of their surface structures (“antigenic drift”), as is the case with seasonal influenza or HIV. Therefore, there is a need for vaccines utilizing the second powerful arm of the adaptive immune system, the cytotoxic defense (1). In this type of immunity, CD8+ T cells specifically recognize peptide fragments derived from (often conserved) pathogen proteins that are presented on the surface of infected cells in the context of MHC I. As a result, the CD8+ T cells become activated, acquire cytotoxic capacity, and eliminate these infected cells in a highly specific fashion.

Apart from prophylactic vaccination, a therapeutic vaccination employing the cytotoxic potential of the immune system is also needed due to the limitations of current therapies against many hematological and solid tumors. In particular, an immune therapy directed at removing residual cancerous tissue or metastases after surgical or chemical ablation of the tumor would be highly desirable.

The repertoire of CD8+ T cells in a naive organism contains only relatively few cells of a given Ag specificity, estimated to be in the order of several hundred cells (2). To induce potent cytotoxicity starting from this low number of Ag-specific T cells, a very effective Ag presentation to CD8+ T cells in an inflammatory context is required. Only dendritic cells (DC) as professional APCs can prime naive CD8+ T cells to become effectors. It is for this reason that elegant strategies to directly introduce Ag into DC in vivo have been developed in the past, originally in the laboratories of Steinman and Nussenzweig (3, 4), and later by others (5–8). All of these approaches to effectively induce cytotoxic CD8+ T cells were based on Ag coupled to Abs directed to surface molecules on DC.

Conventional DC in the mouse are currently being subdivided into two populations. DC dependent in their development on the transcription factor Batf3 are highly capable of presenting exogenous Ag to naive CD8+ T cells (cross-presentation) (9–12), and Batf3-independent DC excel in Ag presentation to CD4+ T cells (10, 13). To optimally induce cytotoxic immunity, specific targeting of Ag into the Batf3-dependent DC is therefore highly desirable. Most of the original targeting studies could not meet this goal, because at that time surface molecules restricted in their expression to this cross-presenting DC subset were not yet known. In consequence, most of the work has been done on targeting Ag via CD205, a C-type lectin receptor present on cross-presenting DC, but also expressed on a variety of cell types in mice (14, 15) and humans (16). Although the desired targeting specificity could not yet be achieved, these studies were pivotal in that they demonstrated that effective CD8+ T cell cytotoxicity could be induced through direct delivery of Ag into DC in vivo.

In the past few years, we and others recognized that the chemokine receptor XCR1 is exclusively expressed on the Batf3-dependent, cross-presenting DC and not elsewhere in the body (17–19). This highly restricted expression of XCR1 in the mouse is mirrored in the human system, where XCR1 can only be found on DC homologous to the mouse cross-presenting subset (18, 20, 21). XCR1 thus appears to be an ideal target to induce cytotoxic immunity in the human.

In the present work, we targeted Ag to XCR1 in the mouse using either a mAb or the specific murine chemokine ligand XCL1. Both vectors delivered Ag into cross-presenting DC in a highly specific fashion and induced, when applied with LPS as adjuvant, potent cytotoxic immunity. Moreover, we generated a transgenic mouse that orthotopically expresses only the human XCR1 receptor on cross-presenting DC. In this model, we could demonstrate efficient induction of cytotoxic immunity with human XCL1 or XCL2 as vectors. The successful targeting of Ag into cross-presenting DC via the human XCR1 under physiological conditions is a major step forward in establishing this system for induction of therapeutic CD8+ T cell cytotoxicity against neoplastic diseases in the human.

Materials and Methods

Mice

C57BL/6 mice (8–10 wk old) were used for experiments and cell isolation, unless indicated otherwise. B6.XCR1-lacZ mice (The Jackson Laboratory) is a strain in which the XCR1 gene has been replaced by the β-Gal reporter gene; these mice were fully backcrossed (>10×) onto the C57BL/6 background. OT-I and OT-II TCR-transgenic mice were crossed onto the B6.PL background to allow identification of CD8+ and CD4+ T cells using the CD90.1 marker. All mice were bred under specific pathogen-free conditions in the animal facility of the Federal Institute for Risk Assessment (Berlin, Germany). Experiments were performed according to state guidelines and approved by the local animal welfare committee.

Generation of huXCR1 transgenic mice

A BAC containing the human XCR1 gene was obtained from Children’s Hospital Oakland Research Institute (clone ID: RP11-527M10). BAC DNA was prepared from bacterial culture grown at 30°C using the Nucleobond BAC 100 kit (Macherey-Nagel), digested with MluI/AscI for removing the bacterial backbone, purified by Sepharose CL-4B chromatography (22), and injected (1–4 ng/μl) into the pronucleus of C57BL/6J fertilized oocytes. Offspring was genotyped using the primer pair 5′-GCACGGTCAAGCTCATCTTCGCC-3′ and 5′-ATGCTCCTTCCAGGCCCGCT-3′. Five transgenic founders (which expressed both wild-type and human XCR1) were crossed with homozygous B6.XCR1-lacZ mice (deficient for XCR1) to obtain mice only expressing human XCR1. The founder line with the highest huXCR1 orthotopic expression was then chosen for further experiments (B6.huXCR1tg).

Cells and cell isolation

Murine splenocytes were obtained by mashing spleens through 70-μm cell sieves into PBS, followed by erythrocyte lysis with ACK buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Where indicated and with lymph nodes (LN), DC were enriched by digesting pieces of lymphoid tissue with collagenase D (500 μg/ml) and DNase I (20 μg/ml; both Roche) in RPMI 1640 containing 2% FCS (low endotoxin; Biochrom) for 20 min at 37°C; EDTA (10 mM) was added for additional 5 min, and cells were filtered through a 70-μm nylon sieve (BD Falcon). Human PBMC and splenocytes were prepared by standard Biocoll (Biochrom) density gradient centrifugation of buffy coats or mashed spleens, respectively. Human splenocytes were from splenectomies in patients suffering from spherocytosis or massive cysts; their use in this study was approved by the local ethics committee.

The DNA coding for full-length murine XCL1 was cloned into the drosophila expression vector pRmHa-3 (23) by standard procedures. The construct for XCL1-StrepTag was designed to contain murine XCL1 and StrepTag (SAWSHPQFEKGGGSGGGSGGGS WSHPQFEK), separated by a glycine-serine linker (GS). The DNA for XCL1-StrepTag was chemically synthesized (Geneart/Life Technologies) and cloned into pRmHa-3. XCL1-OVA was generated by cloning murine XCL1 and full-length chicken OVA, separated by a glycine-serine linker (GS), into pRmHa-3. For generating recombinant MARX10-OVA Ab, we first determined the DNA sequences coding for the complete κ L chain and the V region of the H chain of mAb MARX10. The DNA for the κ L chain was chemically synthesized (Eurofins Genomics) in frame with a DNA sequence coding for a signal peptide and cloned into the mammalian expression vector pTT5 (NRC-Biotechnology Research Institute). The H chain of MARX10-OVA was generated as follows: as a backbone, we used the DNA sequence of DEC-OVA (provided by M.C. Nussenzweig, Rockefeller University, NY) that was cloned via EcoRI/NotI into pTT5. The V region of the MARX10 H chain was chemically synthesized in frame with a sequence for a signal peptide, including an EcoRI restriction site at its 5′ end and a short sequence of the CH1 domain of DEC-OVA, including a unique BstEII restriction site at its 3′ end (Eurofins Genomics). The synthesized DNA fragment was then used to replace the V region of the H chain of the DEC-OVA Ab with that of MARX10 via the restriction sites EcoRI/BstEII. huXCL1-StrepTag was designed to contain DNA for human XCL1 and StrepTag (SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK), separated by a glycine (G). The DNA for huXCL1-StrepTag was chemically synthesized (Eurofins Genomics) and cloned into pRmHa-3. For generating huXCL2-StrepTag, human XCL2 (differing from huXCL1 at positions 28(D→H) and 29(K→R)) was chemically synthesized by Eurofins Genomics and used to replace huXCL1 in the huXCL1-StrepTag plasmid. huXCL1-OVA was generated by fusing human XCL1 via a glycine-serine linker (GS) to full-length chicken OVA, followed by the TEV-cleavage site ENLYFQG (separated by a GS linker) and the StrepTag SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (separated by a GGSGGSG linker). The whole construct was cloned into the pRmHa-3 plasmid. huXCL2 was constructed in an analogous manner.

Expression and purification of recombinant proteins

XCL1, XCL1-StrepTag, huXCL1-StrepTag, huXCL2-StrepTag, XCL1-OVA, huXCL1-OVA, or huXCL2-OVA encoding plasmids were electroporated together with the plasmid phshs.PURO into drosophila Schneider SL-3 cells (24) using a Bio-Rad Gene Pulser (450 V and 500 μF). The phshs.PURO plasmid (provided by M. McKeown, Salk Institute) allows selection of positive transfectants by puromycin. Clones from limiting dilution cultures of transfected SL-3 cells were analyzed for high protein production using either XCL1- or StrepTag-specific ELISA and expanded in serum-free Insect-XPRESS medium (Lonza) on a shaker platform (100 rpm) in normal air at 27°C. XCL1 protein was purified from supernatants by heparin Sepharose affinity chromatography, followed by reversed-phase HPLC; the identity of the protein was verified by mass spectrometry. XCL1-StrepTag protein was purified from the supernatant using HiTrap Heparin HP columns (GE Healthcare). XCL1/2-OVA protein was purified by heparin Sepharose affinity chromatography, followed by anion exchange chromatography on a Mono Q 5/50 GL column (GE Healthcare). huXCL1-StrepTag, huXCL2-StrepTag, and huXCL1-OVA were purified using StrepTrap HP columns (GE Healthcare). pTT5 plasmids encoding the H and L chains of recombinant MARX10-OVA were transiently cotransfected into 293-6E cells (NRC-Biotechnology Research Institute) with linear polyethylenimine. Cells were grown in serum-free FreeStyle F17 expression medium (Life Technologies) supplemented with 0.1% Pluronic F68, 4 mM l-glutamine, and 25 μg/ml G418 on a shaker platform (125 rpm) in 5% CO2 at 37°C. mAb MARX10-OVA was purified from the supernatant using HiTrap Protein G HP columns (GE Healthcare). LPS content of XCL1 (0.26 EU/mg), XCL1-OVA (12 EU/mg used for chemotaxis and test of DC activation in vivo; >50 EU/mg used for in vivo proliferation and CTL induction), and MARX10-OVA (<0.6 EU/mg) was determined using the Endosafe-PTS test (Charles River).

Antibodies

Hybridomas producing mAb recognizing CD4 (clone YTS 191.1), CD8 (53-6.72), CD11c (N418), CD16/32 (2.4G2), CD45R/B220 (RA3-6B2), CD80 (16-10A1), CD86 (GL1), IFN-γ (AN18.17.24), Ly6G/C (RB6-8C5), SIINFEKL-H-2Kb (25-D1.16), and MHC II (M5/114.15.2) were obtained from American Type Culture Collection, and CD90.1 (OX-7) from European Cell Culture Collection. mAb to CD103 (M290) was from BD Biosciences, and to CD69 (H1.2F3) and PD-L2 (TY25) from eBioscience. Anti-CD3 (KT3) was generously provided by H. Savelkoul (University of Wageningen, Wageningen, the Netherlands), anti-CD25 (2E4) by E. Shevach (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD), and anti-CD40 (FGK) by T. Rolink (University of Basel, Basel, Switzerland). Generation of anti-XCR1 mAb MARX10 (19) and anti-ICOS mAb (MIC-280 (25)) has been described before. The nonagonistic mAb MARX10 (mouse IgG2b, in the recombinant version IgG1) does not block the binding of XCL1 to XCR1. For staining of human PBMC, the following Abs were used: CD19 (clone BU12) (26), CD3 (OKT3), CD14 (63D3), and HLA-DR (L243) from American Type Culture Collection; CD11c (BU15), CD16 (3G8), and CD56 (HCD56) from BioLegend; and CD141 (AD5-14H12) from Miltenyi Biotec. mAb D3-13F1 (IgG2b, specific for nitrophenol, a gift of K. Rajewsky) was used as the isotype control for the natural mAb MARX10.

Chemotaxis assay

DC were obtained from digested murine splenic tissue (see cell isolation), and further enriched by centrifugation over a Nycoprep density gradient (1.073 g/ml), followed by positive magnetic sorting with anti-CD11c microbeads (Miltenyi Biotec). For chemotaxis assays, 1 × 106 DC (purity ∼70%) were suspended in 100 μl chemotaxis medium (RPMI 1640, 1% BSA, 50 μM β-ME, 100 μg/ml penicillin/streptomycin) and placed into the upper chamber of a 24-well Transwell system (6.5 mm diameter, 5-μm pore polycarbonate membrane; Corning Costar). The lower chamber was filled with chemotaxis medium to which recombinant XCL1 or XCL1-OVA was added. After incubation in 5% CO2 for 2 h at 37°C, the number of migrated DC was determined by counting cells in the lower chamber using a flow cytometer. DC were identified by staining for XCR1, CD8, CD11c, and MHC II after gating out cells expressing B220 and CD3. The percentage of migrated cells was calculated by dividing the number of cells in the lower chamber by the number of input cells (number migrated cells/number input cells × 100).

In vivo proliferation and in vitro activation of OT-I and OT-II T cells

Recipient mice were adoptively transferred with splenocytes containing 2 × 106 OT-I (CD8+) resting T cells (negative for CD25, CD69, and ICOS), or with 1 × 106 naive transgenic OT-II (CD4+) T cells enriched from splenocytes by positive sorting with CD62L beads (Miltenyi Biotec). For proliferation analysis, OT-I and OT-II cells were labeled with 5 μM CFSE (Invitrogen) before transfer and analyzed 48 h after immunization using the CFSE dilution assay. For measuring IFN-γ production, splenocytes from immunized mice were isolated 72 h after immunization and stimulated with 0.02 μg/ml PMA and 1 μg/ml ionomycin in complete medium for 4.5 h, with 5 μg/ml brefeldin A (all from Sigma-Aldrich) present for the last 3.5 h. Cells were then stained for surface Ags, labeled with 1.34 μM Pacific Orange to mark dead cells for later gating, and fixed with 2% paraformaldehyde. Intracellular staining was performed at RT in a solution of 0.5% saponin (Sigma-Aldrich), 100 μg/ml 2.4G2, and 50 μg/ml purified rat Ig. LPS was removed from OVA using EndoTrap red (Hyglos), resulting in <0.5 EU endotoxin/mg protein as determined by the Endosafe-PTS assay (Charles River).

Determination of OVA-specific Abs in serum

C57BL/6 or homozygous B6.XCR1-lacZ mice were immunized i.v. with OVA (2 mg), MARX10-OVA (0.16 μg), or XCL1-OVA (1 μg), together with 3 μg LPS; controls received only LPS or PBS. After 14 d, OVA-specific Ab in the serum were determined by ELISA. In short, flat-bottom MaxiSorp 96-well immunoplates (Nunc) were coated with 100 μg/ml OVA in PBS overnight at 4°C, blocked with 1% BSA in PBS for 1 h at RT, and incubated with serum dilutions in PBS, 1% BSA for 1 h at 37°C. OVA-specific Ig isotypes were measured with goat anti-mouse IgM-, IgG1-, IgG2a-, and Ig2b-specific Abs coupled to HRP (Southern Biotech Antibodies), and 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich) was used as substrate.

In vivo cytotoxicity assay

Animals were immunized with the indicated amounts of either MARX10-OVA, XCL1-OVA, huXCL1-OVA, or huXCL2-OVA i.v. Six days later, 5 × 106 syngeneic splenocytes were pulsed with SIINFEKL peptide (GenScript) and labeled with 10 μM CFSE (CFSEhigh) in vitro, and 5 × 106 splenocytes were left unpulsed and labeled with 1 μM CFSE (CFSElow). Both preparations were injected together i.v. into immunized and control animals, and the CFSE signal was determined by flow cytometry 18 h later. Specific lysis was calculated using the following formula: specific lysis (%) = 100 − ([CFSEhigh immunized/CFSElow immunized]/[CFSEhigh control/CFSElow control]) × 100.

Tumor model

Six days after immunization, C57BL/6 mice were injected s.c. with 6 × 105 E.G7 cells (EL4 thymoma transfected with soluble OVA) (27) diluted in 50 μl PBS. Because the percentage of E.G7 cells expressing SIINFEKL on the surface decreases over time, the SIINFEKL+ E.G7 cells were flow sorted 3 d before injection using mAb 25-D1.16. Tumor growth was continuously monitored, and tumor size and weight were determined 14 d after E.G7 cell injection.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5. Data are shown as mean or mean ± SEM.

Results

mAb MARX10 specifically binds to the murine chemokine receptor XCR1 (19). To assess whether this mAb can bind to XCR1 in vivo, mAb MARX10 was labeled with Dig and injected i.v. Lymphoid tissues were removed at various time points and analyzed for the presence of the mAb on the surface of cells. mAb MARX10 could be detected on splenic XCR1+ DC, but not on other splenic DC populations, as early as 10 min and as late as 1 wk after injection (Fig. 1A), the latter suggesting a long t1/2 of the mAb in the circulation. Only B cells (0.5%) showed some unspecific staining in the first 4 h, but were later negative (data not shown). No signal was detected on DC in spleens of mice deficient for XCR1 (Fig. 1B). In peripheral LN, MARX10 was found on the surface of most resident XCR1+ DC (MHC IIint), but only on a small population of migratory XCR1+ DC (MHC IIhigh) 14 h after injection (Fig. 1C), indicating that the mAb had good access to lymphoid tissues within the 14-h experimental period, but incomplete access to DC residing in peripheral organs. When splenic tissue sections were analyzed for the presence of mAb MARX10, the pattern obtained (Fig. 1D) was indicative of a highly specific binding to XCR1+ DC (17, 19). Similar studies with the chemokine ligand XCL1 did not give a signal (our unpublished data).

Binding characteristics of mAb MARX10 in vivo. MARX10-Dig (1.25 μg) was injected i.v. into C57BL/6 (A, C, D) or B6.XCR1-lacZ+/+ (B) mice, and controls received a Dig-coupled isotype mAb (A, C) or PBS (D). At the indicated time points, animals were sacrificed and the presence of mAb MARX10 bound to splenocytes was visualized using a fluorophore-coupled anti-Dig reagent in flow cytometry (A–C). To correlate the anti-Dig signal with expression of XCR1, the splenocytes were costained with mAb MARX10-PE in vitro (A–C). Shown are the flow cytometry profiles of splenic (A and B) and skin-draining LN (C) DC. For histological analysis, MARX10-Dig (10 μg) was injected i.v. and splenic tissue was analyzed for bound Ab (blue) 16 h after injection; counterstaining (brown) was with B cell–specific mAb RA3-6B. Scale bar, 100 μm (D). The kinetics experiment was performed once, and all other experiments twice.

Recombinant MARX10-OVA and XCL1-OVA specifically bind to XCR1

To target Ag to cross-presenting DC via XCR1 in vivo, we generated two delivery systems based on mAb MARX10 and XCL1, the specific chemokine ligand for XCR1. In a first step, the Ag-binding regions of mAb MARX10 were grafted onto the backbone of mAb DEC-205, which has previously been point mutated to minimize binding to Fc receptors (3). The chimeric mAb MARX10 was then modified to accommodate OVA as a C-terminal fusion protein (Fig. 2A). Recombinant versions of XCL1 and XCL1-OVA (Fig. 2A, all drawn to scale) were generated in a separate approach. When tested in vitro, both MARX10-OVA and XCL1-OVA specifically bound to XCR1+ DC, but not to DC from XCR1-deficient mice (Fig. 2B), or any other cells (data not shown).

MARX10-OVA and XCL1-OVA specifically bind to XCR1. (A) The V region of the H chain and the V and C regions of the L chain of mAb MARX10 (blue) were grafted onto the backbone of mAb DEC-205 (white), and the entire sequence of OVA (OVA, red) fused to the C terminus of this construct (mAb MARX10-OVA). OVA was also fused to the C terminus of murine XCL1. (B) Splenocytes from C57BL/6 mice (red histograms) or from mice deficient for XCR1 (homozygous B6.XCR1-lacZ mice, filled gray histograms) were stained with MARX10-OVA or XCL1-OVA, and for comparison with the parent molecules MARX10 or XCL1. Shown are the staining profiles after gating on DC defined as CD11c+ MHC II+ Lin− cells. Representative experiment of >10 (MARX10, XCL1) and two (MARX10-OVA, XCL1-OVA).

MARX10-OVA, XCL1-OVA, and XCL1 do not change the activation status of XCR1+ DC in vivo

To test whether recombinant XCL1-OVA is still capable of inducing chemotaxis, Transwell experiments were performed with splenic DC. Various amounts of XCL1-OVA induced chemotaxis of up to 30% of XCR1+ DC, whereas XCR1− DC did not migrate; the functional effects of XCL1-OVA were thus comparable to the parent chemokine XCL1 (Fig. 3A). The targeting vector MARX10-OVA did not induced chemotaxis in analogous experiments (our unpublished data).

Effects of the targeting systems MARX10-OVA and XCL1-OVA on XCR1+ DC. (A) XCL1-OVA, similar to its parent molecule XCL1, selectively induced chemotaxis of XCR1+ DC (migrated % of input, ±SEM) when tested with highly enriched (∼70%) splenic DC in vitro at 10, 100, and 1000 ng/ml (the indicated concentrations are calculated on the content of XCL1 in the reagents). (B) C57BL/6 mice were injected with OVA (2 mg), MARX10-OVA (0.16 μg), XCL1-OVA (1 μg; the indicated mass is calculated on the net content of OVA in the reagents), XCL1 (5 μg), PBS (negative control), or LPS (3 μg, positive control). Fourteen hours later, splenic DC (CD11c+ MHC II+ Lin−) were analyzed for expression levels of CD80, CD86, CD40, and PD-L2, shown as Δ mean fluorescence intensity (MFI; difference between the MFI of the specific Abs and the MFI of the isotype controls). All experiments shown are representative of two independent experiments.

Because XCL1 and XCL1-OVA function as chemoattractants for XCR1+ DC, we assessed whether their use in vivo changes the activation state of XCR1+ DC. MARX10-OVA was also included in this analysis, although this nonblocking mAb does not have agonist activity. MARX10-OVA and XCL1-OVA were injected i.v. at amounts later used for Ag targeting in vivo, XCL1 at higher amounts. Spleens of treated mice were removed 14 h later and analyzed for expression levels of CD80, CD86, CD40, and PD-L2, the most sensitive markers of DC activation. None of the targeting reagents injected changed the expression levels of the activation markers, which remained at the steady state levels observed after injection of PBS (Fig. 3). Even injection of substantial amounts of XCL1, which elicits a short Ca2+ burst after binding to its receptor (17), did not result in a sustained activation of XCR1+ DC in vivo (Fig. 3).

MARX10-OVA and XCL1-OVA induce proliferation of CD4+ T cells, their differentiation to Th1 cells, and the generation of isotype-switched Ag-specific Abs in vivo

XCR1+ DC not only cross-present, but also classically present Ags in the context of MHC II. To examine whether Ag targeted via XCR1 is also processed in the MHC II presentation pathway, C57BL/6 mice were adoptively transferred with naive OT-II CD4+ T cells and injected 1 d later with various doses of OVA, MARX10-OVA, or XCL1-OVA i.v., together with LPS. The frequency of proliferating OT-II CD4+ T cells was determined 48 h later. T cell proliferation could already be observed with 0.005 μg MARX10-OVA (net content of OVA), and maximal proliferation was reached with 0.05 μg (Fig. 4A); MARX10-OVA was thus ∼1000-fold more efficient than nontargeted OVA in this assay (Fig. 4A). XCL1-OVA was ∼10-fold less efficient than MARX10-OVA, but still highly effective as a targeting reagent. When these reagents were tested for their targeting specificity by injection into XCR1-deficient mice (B6.XCR1-lacZ+/+), only MARX10-OVA at 0.5 μg induced some CD4+ T cell proliferation (Fig. 4B). These experiments demonstrated that Ags are specifically targeted to XCR1 and are then efficiently processed and presented in the context of MHC II. To determine whether the activated CD4+ T cells are also polarized in vivo, the transferred OT-II cells were analyzed for the production of various cytokines 72 h after transfer by in vitro stimulation with PMA and ionomycin, followed by intracellular staining. Targeting of Ag into XCR1+ DC in the presence of adjuvant resulted in the secretion of IFN-γ by a substantial proportion of Ag-specific CD4+ T cells (Fig. 4C), whereas IL-4 and IL-10 were not detected (our unpublished data). These results indicated that targeting of Ag via XCR1 in the presence of an adjuvant induces Th1 polarization in Ag-specific CD4+ T cells. Finally, we tested whether the CD4+ T cells acquire helper functions and promote the generation of Ag-specific Abs. To this end, C57BL/6 mice were injected with OVA together with LPS i.v., either in a nontargeted form, or targeted as MARX10-OVA or XCL1-OVA. All three forms of Ag delivery induced substantial levels of IgM, IgG1, IgG2a, and IgG2b OVA-specific Abs, as determined in the serum 14 d later (Fig. 4D).

MARX10-OVA or XCL1-OVA triggers proliferation and Th1 polarization of CD4+ T cells in vivo and induces Ag-specific Ab. C57BL/6 mice were adoptively transferred with 1 × 106 naive, CFSE-labeled OT-II CD4+ T cells. One day later, recipients were injected i.v. with the indicated amounts of soluble OVA, MARX10-OVA, or XCL1-OVA (mass calculated on the net content of OVA) together with LPS (3 μg), and control animals received PBS only. (A) Proliferation of OT-II T cells was determined 48 h after injection of Ag by measuring the percentage of cells that have undergone at least one cell division. (B) Experiment was performed as in (A), but recipients were homozygous B6.XCR1-lacZ mice lacking XCR1. (C) Splenocytes were restimulated 72 h after immunization with PMA and ionomycin for 4.5 h in vitro in the presence of brefeldin A, and IFN-γ was measured by intracellular flow cytometry; shown is the percentage of IFN γ+ OT-II cells. (D) C57BL/6 mice were injected with OVA (2 mg), MARX-OVA (0.16 μg), or XCL1-OVA (1 μg, mass calculated on the net content of OVA) i.v., together with LPS (3 μg); control animals received LPS alone or PBS. Indicated levels of OVA-specific Ab isotypes were determined in serial dilutions of serum by ELISA on day 14 (4 mice per group; shown is the mean; maximal SEM was 0.08). All data shown are representative of at least two independent experiments.

Next, we determined the capacity of the two OVA-targeting systems to induce CD8+ T cell proliferation in vivo. C57BL/6 mice were adoptively transferred with resting OT-I CD8+ T cells and 1 d later injected i.v. with various doses of OVA, MARX10-OVA, or XCL1-OVA, together with LPS. The proportion of OT-I CD8+ T cells that have undergone at least one cell division was determined 48 h later. Minimal CD8+ T cell proliferation could already be observed after application of 0.0016 μg MARX10-OVA, and the maximal signal was reached with ∼0.016–0.05 μg (Fig. 5A). MARX10-OVA was thus at least 200-fold more effective than nontargeted OVA (Fig. 5A). XCL1-OVA was ∼20-fold less efficient than MARX10-OVA, but still ∼10-fold more potent than nontargeted OVA in this assay. The specificity of Ag targeting was assessed by injection of the reagents into homozygous B6.XCR1-lacZ mice. In this study, only higher amounts of MARX10-OVA (0.16 μg) induced proliferation of a proportion of CD8+ T cells, and thus had a similar effect as nontargeted OVA (Fig. 5B). Together, these experiments once more demonstrated that both MARX10-OVA and XCL1-OVA specifically target Ag into XCR1+ DC. At the same time, the results indicated that Ags targeted to XCR1 are efficiently shunted into the MHC I cross-presentation pathway.

MARX10-OVA or XCL1-OVA induces proliferation of CD8+ T cells in vivo. (A) C57BL/6 mice were adoptively transferred with 2 × 106 resting, CFSE-labeled OT-I CD8+ T cells. One day after transfer, recipient animals were injected i.v. with the indicated amounts of soluble OVA, MARX10-OVA, or XCL1-OVA (mass calculated on the net content of OVA) in the presence of LPS (3 μg); control animals received PBS. Proliferation of OT-I T cells was determined 48 h after Ag injection by measuring the percentage of cells that have undergone at least one cell division. (B) Experiment was performed as in (A), but recipients were homozygous B6.XCR1-lacZ animals lacking XCR1. The data shown are representative of two independent experiments.

Induction of CD8+ T cell cytotoxicity was tested in naive mice, without an adoptive transfer of T cells. C57BL/6 mice were injected once with the indicated amounts of OVA, MARX10-OVA, or XCL1-OVA, together with LPS. The degree of induced cytotoxicity was measured 7 d after immunization using an in vivo cytotoxicity assay. Injection of nontargeted OVA with LPS failed to induce cytotoxicity over a large dose range of 30–1000 μg. Only at the highest dose (3000 μg) did nontargeted OVA induce relevant cytotoxicity (∼40% specific lysis; Fig. 6A). In stark contrast, already small amounts of MARX10-OVA (0.05 μg) induced high killing activity (∼60%), and cytotoxicity levels of ∼95–100% were reached by injection of 0.5 μg MARX10-OVA or more (Fig. 6A). Similarly, XCL1-OVA at a dose of 1 μg or more gave in vivo killing activity of up to 95–100% (Fig. 6A). When any of the reagents were injected without LPS, no cytotoxicity was observed (data not shown). To assess the targeting specificity also in the cytotoxicity assay, analogous experiments were performed in B6.XCR1-lacZ+/+ mice. In this study, injection of either MARX10-OVA or XCL1-OVA at the higher dose range did not give any significant cytotoxicity (Fig. 6B). These experiments determined that targeting of Ag into DC via XCR1 is highly effective in inducing CD8+ T cell cytotoxicity, whereas injection of nontargeted OVA is essentially ineffective, even at very high amounts.

MARX10-OVA and XCL1-OVA are highly effective in inducing CD8+ T cell cytotoxicity in vivo. (A) Naive C57BL/6 mice were immunized i.v. once with the indicated amounts of OVA, MARX10-OVA, or XCL1-OVA (mass calculated on the net content of OVA), together with LPS (3 μg). On day 6, the animals were injected with CFSE-labeled target cells to quantitate the induced cytotoxicity in vivo (for details, see Materials and Methods). (B) The experiment was performed as in (A), but treated animals were homozygous B6.XCR1-lacZ mice lacking XCR1. (C) Naive C57BL/6 mice were immunized once with MARX10-OVA (0.16 μg), XCL1-OVA (1 μg), or OVA (1 μg), together with LPS (3 μg) i.v., and control mice were only injected with LPS. Seven days later, mice were inoculated s.c. with 6 × 105 E.G7 cells. On day 14, mice were sacrificed and the weight of the tumor tissue was determined. The data shown are representative of three (A and B) and two (C) independent experiments.

Vaccination with MARX10-OVA or XCL1-OVA prevents tumor outgrowth

To determine whether the cytotoxicity induced through targeting of OVA into XCR1+ DC translates into tumoricidal activity in vivo, naive mice were immunized with MARX10-OVA, XCL1-OVA, or nontargeted OVA, together with LPS. Seven days later, all mice were injected s.c. with a high number (6 × 105) of E.G7 cells, an aggressively growing syngeneic EL4 thymoma transfected with OVA. In control animals and mice injected with nontargeted OVA, the tumor grew quickly to finally reach a size of ∼1 cm in diameter 14 d after inoculation. In clear contrast, all animals immunized with the targeted forms of OVA effectively controlled tumor outgrowth (Fig. 6C). This experiment determined that the induced cytotoxicity is of biological relevance.

Targeting of Ag via human XCR1 induces potent cytotoxicity in vivo

Having demonstrated the efficiency of Ag targeting via XCR1 in the mouse, we sought to determine whether this approach also functions using human XCL1 and its variant XCL2 (differing only at positions 28 and 29) to target human XCR1 under physiological conditions. To this end, we constructed and expressed huXCL1-StrepTag, huXCL2-StrepTag, huXCL1-OVA, and huXCL2-OVA (Fig. 7A). At the same time, we generated mice selectively expressing human XCR1 on cross-presenting DC on a background deficient for murine XCR1 (huXCR1tg). In a first step, binding of the various reagents to human and murine XCR1 was determined. huXCL1 stained 55–80% of CD141+ DC obtained from PBMC or human spleens, but not other DC (Fig. 7B), or other cells (data not shown). Important for subsequent functional experiments, huXCL1 bound to human XCR1 in huXCR1tg mice, but did not react with murine XCR1 in C57BL/6 controls (Fig. 7B). The binding pattern of huXCL2 was indistinguishable from the pattern observed with huXCL1 (Fig. 7B). muXCL1 cross-reacted with human XCR1 in PBMC, spleens, and the huXCR1tg mouse, in which it also detected a XCR1lowMHChigh DC population (Fig. 7B). The targeting constructs containing full-length OVA (huXCL1-OVA, huXCL2-OVA) exhibited a staining pattern comparable to huXCL1 and huXCL2 (our unpublished data).

Human XCR1 can be targeted for induction of potent cytotoxic immunity in vivo. (A) Recombinant fusion proteins huXCL1, huXCL2, huXCL1-OVA, and huXCL2-OVA (drawn to scale). (B) Binding of Strep-tagged huXCL1, huXCL2, and muXCL1 to DC (which were defined both in the human and mouse systems as CD11c+ MHC II+ Lin−) in 1) human PBMC, 2) human spleen, 3) spleens of transgenic mice expressing only human XCR1 on cross-presenting DC (huXCR1tg), and 4) spleens of wild-type C57BL/6 mice; background control (gray histograms) was with Strep-tactin only. (C) Naive huXCR1tg or (D) wild-type C57BL/6 were immunized i.v. with the indicated amounts of huXCL1-OVA or XCL2-OVA (mass based on net content of OVA) together with 3 μg LPS. On day 6, the animals were injected with CFSE-labeled target cells to quantitate the induced cytotoxicity in vivo. The data shown are representative of two independent experiments.

To assess the capacity of human XCL1 or XCL2 to target Ag in vivo, huXCR1tg mice were immunized once with varying amounts of XCL1-OVA or XCL2-OVA i.v., together with LPS. The in vivo cytotoxicity assay on day 6 revealed that 1 μg huXCL1-OVA or 2 μg huXCL2-OVA induced maximal killing activity of 95–100% (Fig. 7C), whereas the same amounts were largely ineffective in wild-type C57BL/6 mice (Fig. 7D). This experiment demonstrated that human XCL1 or XCL2 is capable of efficiently targeting an Ag into cross-presenting DC expressing human XCR1 under physiological conditions.

Discussion

In recent years, the growing understanding of the role of DC subsets in the uptake, processing, and presentation of Ag to CD4+ and CD8+ T cells allowed to define ideal criteria for the design and evaluation of novel vaccines inducing a cytotoxic response. Such a vaccine should do the following: 1) deliver Ags into cross-presenting DC in a highly specific fashion; 2) have no adverse effects on the targeted DC or other cells; 3) allow processing of the delivered Ags in the MHC I pathway; 4) be devoid of any inherently immunogenic components; 5) have a high tissue penetration; 6) provide signals for the differentiation of CD8+ T cells to cytotoxic effectors; and 7) be applicable in humans. We would like to discuss the data presented in this work in view of these criteria.

Targeting specificity

In the mouse, the chemokine receptor XCR1 is exclusively expressed on cross-presenting DC throughout the body. XCR1 is, in fact, the lineage marker for cross-presenting DC in the mouse (19, 28). This restricted expression is in the human mirrored by the exclusive presence of XCR1 on the surface of CD141+ DC, the counterpart of murine cross-presenting DC (18, 20, 21, 29). Although in vivo tests of Ag cross-presentation are not possible in humans, the highly conserved expression and function of XCR1 and its chemokine ligand XCL1/2 strongly indicate that targeting of Ag via XCR1 should lead to efficient cross-presentation also in humans.

For the XCR1-specific mAb MARX10, the targeting specificity could directly be visualized in vivo. As long as injected i.v. at lower, but saturating amounts (1–2 μg), the mAb selectively bound to XCR1+ DC. Fusing the model Ag OVA to either mAb MARX10 or XCL1 did not change their specificity for XCR1. The specificity of Ag delivery could also be documented for both vector systems at a functional level with mice deficient for XCR1.

Currently, only one other molecule is known to be similarly restricted to cross-presenting DC. In humans, Clec9A/DNGR-1 is selectively expressed on CD141+ DC (30, 31); the only report indicating some presence of Clec9A/DNGR-1 on other cell types (32) has to be independently verified yet. Interestingly, the lectin Clec9A/DNGR-1 has been shown to optimize processing of phagocytosed necrotic cells and thus to be an integral part of the cross-presentation machinery (33–36), as is XCR1 (17).

When comparing mAb MARX10 with XCL1 for targeting of Ag, it is apparent that MARX10 binds to XCR1 with higher avidity. Moreover, MARX10 can be detected on the surface of cross-presenting DC still 1 wk after injection, suggesting a relatively long t1/2 in the circulation. Given the higher avidity and the long t1/2, one would expect that the mAb clearly outperforms the chemokine ligand in the delivery of Ag to XCR1. However, when tested for induction of CD8+ T cell cytotoxicity, the difference in efficiency between those two vector systems was surprisingly small in a large series of experiments. Why XCL1 is so efficient in delivering Ag into XCR1+ DC remains unclear at present.

No adverse effects on targeted DC or other cells

The use of the nonblocking, nonagonistic mAb MARX10 demonstrates that delivery of Ag via XCR1 using a mAb can be done without activation or depletion of the targeted DC. The chemokine ligand XCL1 does elicit a short Ca2+ burst upon binding to XCR1 and is an effective chemoattractant (17). Nonetheless, application of high amounts of XCL1 does not induce a sustained activation of DC in vivo, as determined by measuring activation surface molecules. Similarly, no adverse effects of the vector systems are to be expected on nontarget cells, because Abs (as a protein class) and the chemokine XCL1/2 are physiological proteins present in the circulation.

Processing and presentation of delivered Ag in the MHC I pathway

Upon binding of their ligands, chemokine receptors are classically internalized. Therefore, it is not surprising that Ag bound to XCL1, when used as a vector, is efficiently taken up by DC. Interestingly, this is apparently also the case with mAb MARX10, which has no chemokine agonist activity. Because the biochemical pathways involved in cross-presentation of Ag are not well defined, presentation in the MHC I pathway has to be determined empirically. In this respect, XCR1 as a target structure appears to be highly effective, because very low amounts of MARX10-OVA or XCL1-OVA are capable of inducing potent CD8+ cytotoxicity.

In addition to their capacity for Ag cross-presentation, Batf3-dependent DC can also classically process and present Ag in the MHC II pathway. In our experiments, we have observed that targeting of OVA via XCR1 quite efficiently induces activation (our unpublished data), proliferation, and Th1 differentiation of CD4+ T cells, and leads to the generation of Ag-specific Abs. This indicates that Ag internalized by XCR1 is shunted both to the MHC I and MHC II pathways, similar to Ag internalized by Clec9A/DNGR-1 (30, 31).

Absence of inherently immunogenic components

Vaccines usually have to be applied multiple times. It is, therefore, of major importance whether the targeting vector (without attached Ag) is immunogenic. For example, viral vectors typically induce strong immune reactions and memory, and therefore cannot be applied repeatedly. Ample clinical experience indicates that targeting of Ag by humanized or human mAb should not pose such problems. Human XCL1/2 is a physiological protein abundantly secreted into the circulation upon activation of the innate immune system and by activated CD8+ T cells, and therefore tolerance to this protein is fully established. Thus, repeated application of either vector system for targeting of Ag into XCR1+ DC should be effective. This may be quite important in the successive delivery of different Ags using therapeutic vaccination in neoplastic diseases.

Tissue penetration

The relatively high m.w. of Abs prevents their rapid clearance from the circulation, when systemically applied. However, this high m.w. may compromise the tissue penetration of Ab targeting vectors in peripheral lymphoid organs. Our finding of a rather selective detection of MARX10 on resident DC (as opposed to migratory DC) in LN after systemic injection points toward this direction; an engineered smaller Ab format could overcome these potential limitations. XCL1/2, in contrast, is a small protein of ∼10 kDa. As such, this molecule can be expected to have a high tissue penetration, which may only be limited by the size of the fused protein; in the case of (long) peptides, the tissue penetration should remain high.

Induction of CD8+ T cell cytotoxicity

Upon targeting of Ag, we could induce potent CD8+ cytotoxicity when applied together with the adjuvant LPS; in the absence of an adjuvant, only proliferation resulted. This finding is in accordance with other targeting systems (5–8) and indicates that inflammatory signals essential for induction of cytotoxicity have to be provided. In the present work, we used LPS throughout the study, but additional experiments indicate that also other TLR agonists such as CpG or poly(I:C) are fully effective (our unpublished data). Because TLR4, the receptor for LPS, is not present in human cross-presenting DC (21), other adjuvants such as poly(I:C) will have to be applied in the human.

Applicability in humans

We could demonstrate that human XCL1/2-OVA induces potent CD8+ cytotoxicity when injected into mice expressing only human XCR1 on their cross-presenting DC. This finding demonstrates that huXCL1/2 in vivo binds to human XCR1 with sufficient avidity to be used as a targeting system. Furthermore, these results indicate a rapid internalization of human XCR1, followed by an efficient Ag processing and presentation in the MHC I pathway. Because one can assume that cross-presenting human DC process and present Ag with the same efficiency as their mouse counterparts, these results strongly indicate that targeting of Ag to XCR1 is a viable approach for the induction of cytotoxicity in humans. In the next step, this has to be demonstrated in nonhuman primates.

Disclosures

R.A.K. is inventor of a patent application by the Robert Koch-Institute on targeting XCR1 for vaccination.

Acknowledgments

We thank Michel C. Nussenzweig for the generous gift of the cDNA of mAb DEC-205.

Footnotes

This work was supported by grants of the Wilhelm Sander Foundation, the Fritz Thyssen Foundation, the Validation of Innovation program of the Bundesministerium für Bildung und Forschung, and in part by Deutsche Forschungsgemeinschaft grants to R.A.K. (Kr 827/16-1 and Kr 827/18-1).

. 1995. Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145. I. Expression on dendritic cells and other subsets of mouse leukocytes.Cell. Immunol.163: 148–156.

. 2014. Ontogenic, phenotypic, and functional characterization of XCR1+ dendritic cells leads to a consistent classification of intestinal dendritic cells based on the expression of XCR1 and SIRPα.Front. Immunol.5: 326.